Project Summary New tools for large-scale recording of neuronal activity in a living and behaving brain are essential for a better understanding of brain function, efficient analysis and treatment of neuronal disorders. Time resolved volumetric photo-acoustic imaging offers tremendous potential for large-scale brain recording due to its exquisite penetration into living tissues. Most recent developments in instrumentation for photo-acoustic neuroimaging are rapidly advancing the field with ever increasing resolution, sensitivity, field of view and frame rates. Yet, these developments need to be matched by concomitant engineering of suitable contrast agents and activity reporters. The existing genetically encoded calcium indicators have shown promise in proof-of-principle photoacoustic studies but their absorbance maxima in the blue-green ranges of the spectrum is vastly interfered by the strong absorption of hemoglobin in mammalian brains. On the contrary, absorbance of bacterial phytochrome ideally ranges into the near infrared. This project will engineer fluorescent proteins based on bacterial phytochrome into powerful calcium sensors for photo-acoustics. We will insert small calcium binding domains into bacterial phytochromes either by using existing structural information or by using random insertion strategies to turn them into efficient absorbance-based calcium reporters. Prototypical reporters will then be further engineered using diversification and large scale screening of variants using a unique new automated robotic screening station developed by the applicants. Finally, high performing sensor variants will be validated in mouse visual cortex in vivo. Thus, the proposal combines complementary expertise in development of photo-acoustic instrumentation and large scale biosensor engineering to enable fast volumetric activity imaging of the intact scattering mammalian brain.